BM是由英国癌症研究中心博士助学金的支持。公司是由生命科学科研计算机网威尔士博士助学金的支持。 VB由癌症研究威尔士博士助学金的支持。 DKC是威康信托基金研究职业发展研究员（WT095767）。 AKS是威康信托基金研究员。 GHM通过接头生命科学研究网络威尔士和Tenovus癌症护理博士助学金资助。我们感谢在钻石光源的员工提供便利和支持。

1.蛋白表达<ol><li>使基因构建为可溶性的TCR和pHLAs的产生，如先前17,18进行详细说明。设计一个5&#39;BamH1位和3&#39;EcoRⅠ小限制位点用于插入pGMT7向量中的每个构造。 </li><li>转化大肠杆菌 Rosetta（DE3）pLysS中含有通过孵育50-200纳克/微升质粒与5微升大肠杆菌 5分钟的1微升编码感兴趣的蛋白质的序列的pGMT7衍生质粒载体在4℃下，2分钟，在42℃，并在4℃下5分钟，并在37℃下在补充有50毫克/升羧苄青霉素的LB琼脂平板过夜积垢。 </li><li>挑选单个菌落并在补充有100μM的羧苄青霉素，直到该悬浮液在37℃和220rpm下在30 TYP介质中的溶液（16克/升胰蛋白胨16克/升酵母提取物，和5克/升HK 2 O 4）生长到达光德nsity 0.4和0.6之间（OD 600）。 </li><li>通过3小时引入0.5毫异丙β-D-1-硫代半乳糖苷（IPTG）诱导蛋白质产生在5毫升等分试样。保持悬浮液的20μL的使用和不使用诱导十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）进行分析和染色凝胶。 </li><li>添加起子培养至1L TYP补充有100μM的羧苄青霉素和如上面步骤1.3中所述，直至该悬浮液达到0.4和0.6之间的OD 600生长的细胞。 </li><li>诱导蛋白质表达为用0.5mM IPTG 3小时。离心20分钟将细胞以3000 xg离心，小心地倒出上清液。 </li><li>溶解在40ml裂解缓冲液的沉淀（10毫摩尔Tris，pH值8.1; 10mM氯化镁， 氯化镁 ; 150毫摩尔NaCl; 10％甘油）中，在冰上超声处理采用2秒的时间间隔30分钟，在60％的功率，并在室温（RT）下温育30分钟，0.1克/升的DNase。 </li><li>对待SUS含有包涵体（IB）用100mL洗涤缓冲液的形式蛋白质退休金（0.5％的Triton X-100; 50毫摩尔Tris，pH值8.1; 100毫摩尔NaCl;和10mM EDTA）中。 </li><li>离心样品在4℃下20分钟，8000 XG，小心地倒出上清液。重悬在100毫升再悬浮缓冲液的沉淀（50毫摩尔Tris，pH值8.1; 100毫摩尔NaCl; 10毫摩尔EDTA，pH值8.1），离心机如之前在8000 xg离心，并小心地倒出上清液。 </li><li>最后，溶解沉淀在10mL胍缓冲液（6M胍; 50毫摩尔Tris，pH值8.1; 2mM EDTA的，pH值8.1;和100mM NaCl）中，并用分光光度计测量280nm处的蛋白浓度。 </li></ol> 2. PHLA和TCR复性<ol><li>为PHLA refolds，（用生物素标记或HLA-A2），在37℃的水浴中在最终体积混合30毫克的HLA-A2的的IB，30毫克β2M的IB，以及30分钟4毫克肽的6毫升胍缓冲液中补充了10毫二硫苏糖醇醇（DTT）。 </li><li>通过在1升预先冷却的HLA再折叠缓冲液中稀释之前的混合引发蛋白复性（50毫摩尔Tris，pH值8.1; 400 mM L-精氨酸; 2mM EDTA的，pH值8.1; 6毫半胱胺;和4mM胱胺）。 </li><li>离开的HLA重折叠在4℃下搅拌3小时，然后将其转移到一个12.4 kDa的MWCO（分子量截止）透析管和用于对20升的10mM Tris，pH值8.1的24小时透析两次。 </li><li>对于TCR refolds，30分钟，在37℃的水浴中在6毫升补充有10毫摩尔DTT胍缓冲液中混合30毫克的TCRα链的IB和30毫克的TCRβ链的IB的。 </li><li>通过在1升预先冷却的TCR再折叠缓冲液中稀释变性的TCR混合物发起蛋白复性（50毫摩尔Tris，pH值8.1; 2.5M尿素; 2毫摩尔EDTA，pH值8.1; 6毫半胱胺;和4mM胱胺）3 H。 </li><li>转移到再折叠12.4 kDa的截留分子量透析管和24小时透析两次对20升10毫米的Tris，pH值8.1。 </li><li>同时过滤掉了PHLA或TCR REF使用用于纯化步骤0.45微米的膜过滤器的年轻人。 </li></ol> 3.纯化通过快速蛋白质液相层析（FPLC） <ol><li>加载过滤再折叠制备（无论PHLA或TCR）到7.9毫升，50微米阴离子交换树脂柱预平衡用20ml的10mM Tris，柔性和直观的色谱系统上的pH 8.1的。 </li><li>洗脱蛋白在含有盐梯度5毫升/分钟（0-500毫摩尔NaCl在10mM的Tris，pH值8.1，用8个柱体积），并收集1毫升的级分。 </li><li>分析对应于通过SDS-PAGE感兴趣的蛋白质的级分，汇集含有目的蛋白质一起的级分，并在4000 xg离心20分钟，离心浓缩下来到500μL10-kDa的截留分子量20或10 kDa的MWCO 4 ，丢弃流过。 </li><li>加载浓缩蛋白制剂到2mL注射环到24毫升尺寸排阻层析柱与该应用预平衡ropriate洗脱缓冲液:磷酸盐缓冲盐水（PBS），HBS（10mM的HEPES，pH值7.4; 150毫摩尔NaCl; 3.4毫摩尔EDTA和0.005％表面活性剂），或晶体缓冲液（10mM的Tris，pH值8.1，和10mM NaCl的）。 </li><li>洗脱以0.5毫升/分钟以上1柱体积的流率的蛋白质;收集含的通过SDS-PAGE验证感兴趣的蛋白1毫升的级分。 注意:这些方法被用于产生可溶性PMEL17 TCR和的gp100的TCR，以及所有在本研究中使用的pHLAs的HLA-A * 0201与YLEPGPVTA（A2-YLE），YLEPGPVTV（A2-YLE-9V），ALEPGPVTA （A2-YLE-1A），YLAPGPVTA（A2-YLE-3A），YLEAGPVTA（A2-YLE-4A），YLEPAPVTA（A2-YLE-5A），YLEPGAVTA（A2-YLE-6A），YLEPGPATA（A2-YLE-图7A），或YLEPGPVAA（A2-YLE-8A）。 </li></ol> 4，表面等离子体共振（SPR）分析<ol><li>执行使用装备有在CM5传感器芯片19中的分子间相互作用分析系统平衡结合分析或热力学分析。 </li><li>通过激活F中的CM5芯片降脂1:100毫N-羟基琥珀酰亚胺（NHS）和400mM的1-乙基-3-（3-二甲基丙基）-carboiimide（EDC）的10分钟1混合，以10微升/分钟的流速，在25℃ C。 </li><li>负载大约5000响应单位（RU）链霉的（200微克的110微升/毫升10mM醋酸，pH4.5）中通过共价键在所有四个流动细胞连结至芯片表面，并使用1M乙醇胺盐酸100微升停用任何剩余的反应性基团。 </li><li> PHLA的夫妇约500-600位，在以10微升/分钟的缓慢流速〜1μM的商业缓冲液（由生产商提供），向CM5传感芯片，以确保在芯片表面上均匀分布。 </li><li>饱和，在商业缓冲液为1mM生物素（由生产商提供的）为60秒的芯片表面。 </li><li>注入在相关流动细胞可溶性TCR的十个连续稀释在30微升/分钟，在25℃的高流速。 </li><li>计算平衡结合常数（K D（E））使用非线性曲线拟合（γ=（P1X）/（P 2 + x）的）20个值。 注:Y =响应单位，X =分析师浓度，P1 = R 最大 P2 = 杀敌 。 </li><li>进行动力学分析假定1:1 Langmuir结合并在软件包2使用全局拟合算法拟合数据。 </li><li> 5℃，12℃，18℃，25℃，和30℃19:通过在以下温度重复该方法执行热力学实验。 </li><li>使用通过SPR确定在K D取值在不同的温度来计算ΔG°使用标准热力学等式（ΔG°= -RTlnK D）19。 注:R =气体常数，T =温度以K，LN =自然对数。 </li><li> 19 -根据吉布斯-亥姆霍兹方程（TΔS°ΔG°=△H）计算热力学参数。 </LI&gt; <li>积使用非线性回归拟合三参数方程（结合自由能，ΔG°（ΔG°= -RTlnK D）中 ，对温度（K）Y =ΔH+ΔCp*（X-298）-x *ΔS- X *ΔCp* LN（X / 298））19。 注:Y =温度K，X =ΔG°。 </li></ol> 5.温滴定量热（ITC） <ol><li>执行使用等温滴定热量ITC实验。注入30μMPHLA进入热量计单元和负载210μM的可溶性TCR到注射器。使用下面的缓冲液条件:20毫摩尔的Hepes含有150mM NaCl的（pH 7.4）中。 </li><li>执行20 TCR注射，每次2μL体积。使用分析软件计算出ΔH和杀敌 。 </li></ol> 6.结晶，衍射数据收集和模型修正<ol><li>执行使用结晶机器人结晶试验。 </li><li>通过生长晶体VAP或者通过与含有60微升结晶缓冲液（母液）21的储存器96孔的板中的坐滴技术扩散在18℃。 </li><li>通过在10-kDa的分子量截断离心浓缩以3000 xg离心纺丝浓缩可溶性PHLA在晶体缓冲器大约10毫克/毫升（0.2毫摩尔）。 </li><li>用于共结构复杂，混合TCR和PHLA在1:1的摩尔比在大约10毫克/毫升（0.1毫摩尔）得到的蛋白质溶液。 </li><li>添加200 NL PHLA的单独，或1:TCR和PHLA的1摩尔比混合，以从结晶屏幕使用结晶机器人每个储存溶液的200 nL和得分为结晶在显微镜下24小时，48小时，72后小时，然后每周一次。 </li><li>通过浸没并将它们存储在液氮温度（100 K）收获单晶通过手动将它们安装在冷冻循环的显微镜和下低温冷却它们。 注:加载晶体需要一点PRAC的蒂斯，并决定哪些晶体数据收集不够好来自经验。作为一个经验法则，更大和更经常晶体，效果更佳。 </li><li>在100 K。氮气流收集数据 注:此数据是在钻石光源（DLS）英国国家同步的科学设施收购。 </li><li>通过使用这两个MOSFLM 22估计与xia2的反射强度分析数据和XDS软件包23，然后扩展与SCALA或无目的24和CCP4软件包25中的数据。 </li><li>解决使用PHASER 26分子置换的结构。 </li><li>调整与COOT 27的模型和REFMAC5 28细化模型。 </li><li>准备图形表示与PYMOL 29。 </li><li>通过"接触"计算的接触计划在CCP4包。使用4截止的范德华接触和3.4截止的氢键和盐桥。 </li><li>计算出CCP4包使用"SC"节目表面的互补性。 </li><li>计算TCR-PHLA复杂的交叉角，如上所述30。 注:在本研究中，反射数据和最终的模型坐标分别与PDB数据库沉积（PMEL17 TCR-A2-YLE-9V PDB:5EU6，A2-YLE PDB:5EU3，A2-YLE-3A PDB:5EU4和A2 -YLE-5A PDB:5EU5）。 </li></ol>

The authors have no conflicts of interest or competing financial interests.

这里所概述的协议提供的T细胞应答中的任何人的疾病的背景下的分子和细胞解剖的框架。虽然癌症是本研究的主要焦点，我们使用了非常类似的方法来研究病毒32，33，34，35，T细胞反应36，37和38自身免疫，39，40中。此外，我们已使用这些技术更广泛地理解支配T-细胞抗原识别2，19，41，42中的分子原理。事实上，修改的不可预测性的肽残基，甚至是那些的T细胞受体接触残基之外，影响T细胞识别有heteroclitic肽的设计具有重要意义。这些发现直接促成新型的T细胞疗法的发展，包括多肽疫苗6,43和人工高亲和力的TCR 3，4，5，20，44，以及增强诊断45，46，47。 该协议中的关键步骤 高纯度的，功能性蛋白质的产生是所有的在本文中概述的方法是至关重要的。 修改和故障排除 在产生高纯度蛋白的困难往往涉及到高度表达制纯净，不溶性的IB从大肠杆菌表达系统。通常，修饰的表达协议（ 例如，诱导在不同的光密度，使用不同的大肠杆菌菌株，或者使用不同的介质形成）可以解决这些问题。 该技术的局限性 这些技术使用那些在细胞表面正常表达可溶性蛋白分子（TCR和PHLA）。因此，重要的是要确保结构/生物物理研究结果与蜂窝的方法来确认生物学意义一致。 相对于现有的/替代方法的技术意义 通过使用X-射线晶体学和通过功能分析证实生物物理学，我们和其他人已证明的TCR特异性的癌症抗原表位通常具有低结合亲和性48。这种低TCR亲和力可能有助于解释为什么T细胞ARË在清除癌细胞不能自然有效。抗癌的TCR和同源肿瘤抗原之间复合物的高分辨率原子结构已经开始揭示这个弱亲和力的分子基础。此外，这些研究是用于确定所依据设计来克服这个问题的治疗性干预的机制，播种未来改进16有帮助的。在这项研究中，我们检查了一个天然存在的αβTCR的第一结构中的复杂带的gp100 HLA-A * 0201限制性黑素瘤表位。的结构，与进行了深入的生物物理检查结合，揭示了相互作用的整体结合模式。我们还发现了意想不到的分子开关，从而发生的肽的突变形式，即废除TCR结合和CD8 + T细胞识别（功能实验）（使用表面等离子体共振评估）。这是唯一可能证明HLA抗原presentati的新机制使用所描述的高分辨率方法。 掌握这一技术后，未来的应用或指示 总体而言，当与鲁棒功能分析相结合我们的结果表明X-射线结晶学和生物物理方法的力量。使用这些方法，有可能以剖析出支配T-细胞抗原识别精确的分子机制。实际上，它也可以用这种方式来解决未连接的TCR的结构中，展示的构象变化如何抗原歧视49，50，51中发挥作用。更好地理解的高度复杂性和动态性的支撑TCR-PHLA相互作用也有治疗的设计明显的影响。能够直接"看"正在治疗靶向的修改对抗原识别中的分子，以及效果N，显然会提高这些药物的向前发展。在这项研究中，我们表明，即使改变在单个肽残未很大程度上由TCR接合可以不可预知发送到在与HLA结合的肽，其中，反过来，大幅改变的T细胞识别其它残基的结构变化。设计未来的疗法的广泛的人类疾病时T细胞抗原识别中所采用的分子机制的更完整的理解将是非常有益的。

X射线晶体学已经，并且将继续是一个非常强大的技术来理解配体 - 受体相互作用的性质。通过可视化的原子细节这些相互作用，不仅是有可能泄露理事许多生物过程的分子机制，但它也可以直接改变为治疗益处接触界面。加之技术如表面等离子体共振和等温滴定量热法（仅举几），这样的修改可以被生物物理分析，以评估结合亲和力相互作用动力学和热力学的直接影响。最后，通过在相关的细胞类型执行功能实验中，修饰的受体 - 配体相互作用的分子和功能的影响的详细图像可以收集，提供非常具体的机械信息。总体而言，这些类型的方法提供的原子分辨率图像使阻止<html>的生物系统如何工作，具有诊断和治疗的进步随之而来的影响mination。 我们实验室常规使用这些技术来研究介导人T细胞免疫病原体和癌症，自身免疫的受体，和移植中。这里，我们专注于人类CD8 + T细胞应答于癌症，由T细胞受体（TCR）和人白细胞抗原（HLA）-restricted肿瘤衍生肽（PHLA）之间的相互作用介导的。因为，虽然CD8 + T细胞能够靶向癌细胞，我们和其他人以前曾表明，抗癌的TCR次优结合其同源PHLA 1,2这是很重要的。因此，许多实验室已尝试改变任TCR 3，4，5或肽配位体6，类=以增加免疫原性，并更好地靶癌细胞"外部参照"&gt; 7,8。然而，这些方法并不总是有效，并且可以具有严重的副作用，包括脱靶毒性4，9，10。进一步的研究探讨支配癌抗原的T细胞识别的分子机制将是克服这些缺点是至关重要的。 在本研究中，我们侧重于针对由特定的分化黑色素细胞抗原的一个片段CD8 + T细胞自体的黑色素瘤细胞的响应糖蛋白100（的gp100）的gp100 280-288，由HLA-A * 0201（最常提出-expressed人PHLA I类）。该抗原已黑素瘤免疫治疗的广泛研究的目标，并已发展为一个所谓的"heteroclitic"肽，其中一个缬氨酸取代丙氨酸在锚位9，以提高稳定性PHLA 11。这种方法被用来增强体外黑素瘤-反应性CTL的诱导，并已在临床试验中12被成功地使用。然而，修改的肽残基可以具有对T细胞特异性，通过最heterolitic肽的疗效不佳在诊所6,13展示了不可预测的影响。实际上，的gp100 280-288的另一个heteroclitic形式，其中肽残Glu3代替丙氨酸，通过的gp100 280-288特异性T细胞14，15的多克隆群体废止认可。之前我们已经证实，即使在肽锚残留微小的变化能极大地改变不可预知的方式6，16的T细胞识别。因此，研究的重点是构建ING的CD8 + T细胞是如何识别的TCR和PHLA之间的相互作用的gp100以及如何修改的更详细的图像可能会影响该功能。 在这里，我们产生的具体由HLA-A * 0201（A2-YLE）颁发的gp100 280-288 2电阻温度系数的高纯度，可溶性形式，以及PHLA的自然和改变形式。这些试剂被用来产生蛋白质晶体使用A2-YLE的heteroclitic形式，以及两个在未连接形式的突变体pHLAs来解决人类的TCR的三元原子结构复杂。然后，我们使用的肽扫描方法，通过进行深入的生物物理实验，以证明肽取代的上的TCR的影响。最后，我们产生了转基因CD8 + T细胞系，重新编程以表达A2-YLE特异性的TCR的一个，以执行功能性实验来测试各种肽修饰的生物学影响。这些数据表明，即使莫迪fications的肽是T细胞受体结合基序之外的残基可具有不可预测的连锁上废除TCR结合和T细胞识别相邻的肽残基的影响。我们的研究结果表示底层黑素瘤此重要的治疗目标的T细胞识别的结构机制的第一个例子。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>S.O.C. media</td>
			<td>Invitrogen</td>
		</tr>
		<tr>
			<td>Orbi-Safe New Orbit incubator</td>
			<td>Sanyo</td>
		</tr>
		<tr>
			<td>carbenicillin&#160;</td>
			<td>Sigma</td>
		</tr>
		<tr>
			<td>IPTG&#160;</td>
			<td>Fisher</td>
		</tr>
		<tr>
			<td>Quick Coomassie Stain SafeStain&#160;</td>
			<td>Generon</td>
		</tr>
		<tr>
			<td>Legend RT centrifuge&#160;</td>
			<td>Sorvall</td>
		</tr>
		<tr>
			<td>Tris&#160;</td>
			<td>Fisher</td>
		</tr>
		<tr>
			<td>magnesium chloride&#160;</td>
			<td>Arcos</td>
		</tr>
		<tr>
			<td>NaCl&#160;</td>
			<td>Fisher</td>
		</tr>
		<tr>
			<td>glycerol&#160;</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Sonopuls HD 2070&#160;</td>
			<td>Bandelin</td>
		</tr>
		<tr>
			<td>DNAse&#160;</td>
			<td>Fisher</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>EDTA&#160;</td>
			<td>Fisher</td>
		</tr>
		<tr>
			<td>guanidine&#160;</td>
			<td>Fisher</td>
		</tr>
		<tr>
			<td>NanoDrop ND1000&#160;</td>
			<td>Thermo</td>
		</tr>
		<tr>
			<td>Peptides</td>
			<td>Peptide Protein Research</td>
		</tr>
		<tr>
			<td>dithiothreitol&#160;</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>L-arginine&#160;</td>
			<td>SAFC</td>
		</tr>
		<tr>
			<td>cysteamine&#160;</td>
			<td>Fisher</td>
		</tr>
		<tr>
			<td>cystamine&#160;</td>
			<td>Fisher</td>
		</tr>
		<tr>
			<td>dialysis tubing&#160;</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>urea&#160;</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Metricel 0.45 &#956;m membrane filter</td>
			<td>Pall</td>
		</tr>
		<tr>
			<td>POROS 10/100 HQ&#160;</td>
			<td>Applied biosystems</td>
		</tr>
		<tr>
			<td>&#196;KTA pure FPLC system&#160;</td>
			<td>GE Healthcare Life Sciences</td>
		</tr>
		<tr>
			<td>10 kDa MWCO Vivaspin 20&#160;</td>
			<td>Sartorius</td>
		</tr>
		<tr>
			<td>10 kDa MWCO Vivaspin 4&#160;</td>
			<td>Sartorius</td>
		</tr>
		<tr>
			<td>Superdex 200 10/300 GL column&#160;</td>
			<td>GE Healthcare Life Sciences</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline&#160;</td>
			<td>Oxoid</td>
		</tr>
		<tr>
			<td>BIAcore buffer HBS&#160;</td>
			<td>GE Healthcare Life Sciences</td>
		</tr>
		<tr>
			<td>Biotinylation kit</td>
			<td>Avidity</td>
		</tr>
		<tr>
			<td>BIAcore T200</td>
			<td>GE Healthcare Life Sciences</td>
		</tr>
		<tr>
			<td>CM5 chip coupling kit</td>
			<td>GE Healthcare Life Sciences</td>
		</tr>
		<tr>
			<td>streptavidin&#160;</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Microcal VP-ITC&#160;</td>
			<td>GE Healthcare Life Sciences</td>
		</tr>
		<tr>
			<td>Hepes&#160;</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Gryphon crystallisation robot</td>
			<td>Art Robbins Instruments</td>
		</tr>
		<tr>
			<td>Intelli-plate&#160;</td>
			<td>Art Robbins Instruments</td>
		</tr>
		<tr>
			<td>Crystallisation screens and reagents</td>
			<td>Molecular Dimensions</td>
		</tr>
		<tr>
			<td>75 cm2 flask&#160;</td>
			<td>Greiner Bio-One</td>
		</tr>
		<tr>
			<td>0.22 &#956;m filters</td>
			<td>Miller-GP, Millipore</td>
		</tr>
		<tr>
			<td>Ultra-Clear ultracentrifuge tube&#160;</td>
			<td>Beckman Coulter</td>
		</tr>
		<tr>
			<td>Optima L-100 XP with SW28 rotor</td>
			<td>Beckman Coulter</td>
		</tr>
		<tr>
			<td>CD8 microbeads&#160;</td>
			<td>Miltenyi Biotec</td>
		</tr>
		<tr>
			<td>anti-CD3/CD28 Dynabeads&#160;</td>
			<td>Invitrogen</td>
		</tr>
		<tr>
			<td>anti-PE microbeads&#160;</td>
			<td>Miltenyi Biotec</td>
		</tr>
		<tr>
			<td>CK medium, PHA</td>
			<td>Alere Ltd</td>
		</tr>
		<tr>
			<td>anti-TCRV&#946; mAb&#160;</td>
			<td>BD</td>
		</tr>
		<tr>
			<td>dasatinib&#160;</td>
			<td>Axon</td>
		</tr>
		<tr>
			<td>MIP-1&#946; and TNF&#945; ELISA&#160;</td>
			<td>R&#38;D Systems</td>
		</tr>
		<tr>
			<td>&#160;51Cr&#160;</td>
			<td>PerkinElmer</td>
		</tr>
		<tr>
			<td>1450 Microbeta counter</td>
			<td>PerkinElmer</td>
		</tr>
	</tbody>
</table>

using x-ray crystallography, biophysics, and functional assays to determine the mechanisms governing t-cell receptor recognition of cancer antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

采用上述方法，我们产生可溶性TCR（ 表1）和PHLA分子由CD8 + T细胞的深入进行的gp100 280-288识别分子分析。的改性大肠杆菌表达系统用于生成不溶性的IB为两者的TCR（α和β链）和pHLAs（α链和β2M）的每个单独的链。这种方法具有相对廉价和容易建立优势，并产生蛋白质的产量大（100-500毫克/升的文化）。此外，如果保存在-80℃下的不溶性蛋白质是高度稳定的。然后，我们使用一个完善的重折叠和纯化技术产生功能性的，同质的，可溶性蛋白。此方法是用于产生蛋白质为生物物理，结构，和细胞的实验中，以及可用于诊断或治疗试剂是有用的。 表2）结合亲和力。该实验结果显示其残留肽是最重要的TCR结合。使用这种技术的结合亲和力的高分辨率分析是用于确定控制蛋白质 - 蛋白质相互作用的生物学机制，用于分析治疗分子的结合亲和力是非常有用的，以及。 然后，我们在复杂结晶的特定黑素瘤可溶性TCR（PMEL17 TCR）具有修饰的肿瘤衍生PHLA（A2-YLE-9V）调查在原子分辨率（ 图1和2和表3）的结合模式。这些实验提供了两个分子之间的结合界面的直接可视化，providin摹关于执政的相互作用的基本原则的关键信息。我们进一步进行同时使用SPR和ITC的相互作用的热力学分析，揭示了使结合（ 图3）作出积极贡献。这些分析是由两种蛋白质（ 图4和表4）之间的接触印迹的高分辨率描述进一步支持。 然后，我们解决了未连接PHLA分子的结构，呈现肽的突变形式，揭示了一种分子开关可以解释为什么废除TCR结合某些突变（ 图5）。 总体而言，这些技术提供了新的数据证明该机制解释T细胞如何识别黑素瘤衍生的抗原是抗癌疗法的一个重要目标。更广泛地说，这些技术可用于研究几乎任何受体 - 配体相互作用，揭示可能被靶向用于新的治疗进展的新的生物机制。 <img alt="图1" src="/files/ftp_upload/54991/54991fig1.jpg" /> 图1:密度图分析。左栏显示了省略，其中该模型是在不存在肽的精制地图。差密度在3.0西格马轮廓，正轮廓示于绿色和负轮廓是红色的。右手栏示出了在1.0西格马随后细化使用由REFMAC5施加自动非晶体学对称约束后所观察到的图（示出为围绕蛋白质链的棒表示灰色目）。 （ 一 ）PMEL17 TCR-A2-YLE-9V与TCR CDR3模型循环色蓝（α链）和橙色（β链）和绿色的肽。 （B）型号为A2-与YLE肽呈深绿色。 （℃）A2-YLE-3A与肽橙色的型号（A2-YLE-3A，有在不对称单位2的分子，但这些实际上是相同的省略和密度图的方面，因此，只复制1此处示出）。 （D）为A2-YLE-5A与肽涂上粉红色的模型。从31参考授权转载。 <a href="http://ecsource.jove.com/files/ftp_upload/54991/54991fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54991/54991fig2.jpg" /> 图2:PMEL17的TCR与A2-YLE-9V复杂的概述。在PMEL17 TCR-A2-YLE-9V复杂的（A）卡通表示。该TCR颜色为黑色; TCR CDR环显示（红，CDR1α;深绿色，CDR2α，蓝色，CDR3α;黄色，CDR1β;浅绿色，CDR2 ^6 ;;橙色，CDR3β）;与HLA-A * 0201以灰色示出。该YLE-9V肽是由青枝表示。在PMEL17 TCR CDR的残基（B）表面，并坚持表示回路（颜色编码为A），使联系A2-YLE表面（A2，灰色; YLE-9V，青枝）。黑斜线指示TCR的相对于所述YLEPGPVTV肽（46.15°）的长轴交叉角。 （ 三 ）PMEL17 TCR的A2-YLE-9V表面上（A2，灰色）的联系足迹;紫色和绿色（表面和棍棒）表示HLA-A * 0201和YLE残留物，分别由GP100 TCR接触。截止3.4的氢键和4对于范德华联系。经许可后转载自参考31. <a href="http://ecsource.jove.com/files/ftp_upload/54991/54991fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alT ="图3"SRC ="/文件/ ftp_upload / 54991 / 54991fig3.jpg"/&gt; 图3:PMEL17 TCR-A2-YLE的相互作用的热力学分析。 （A）PMEL17 TCR均衡结合在5至A2-YLE响应，12，18，25和37℃跨越九至十TCR系列稀释。 SPR生和拟合数据（假设为1:1 Langmuir结合）在每个曲线的插图中示出，并用于使用全局拟合算法（BIA评估3.1） 来计算和K off值为ķ。该表显示平衡结合（K D（E））和动力学结合常数（K D（K）= K 关闭 / K 于 ）在每个温度。的平衡结合常数（K D，μM）利用非线性拟合计算的值（Y =（P1X）/（P 2 + X））。 （B）中的热力学参数是根据吉布斯-亥姆霍兹方程计算（ΔG°=ΔH° - TΔS°）。的结合自由能，ΔG°（ΔG°= -RTlnK D），反对温度作图（使用非线性回归拟合三参数方程K）（Y =ΔH°+ΔCp°*（X-298）-X *ΔS°-x *ΔCp° * LN（X / 298））。焓（ΔH°）和熵（TΔS°）在298K（25℃）示于千卡/摩尔和温度（K）的非线性回归计算作图的自由能（△G°）。 （C）等温量热滴定法（ITC）的三围为PMEL17 TCR-A2-YLE互动。焓（ΔH°）和熵（TΔS°）在298K（25℃）示于千卡/摩尔。从31参考授权转载。 <a href="http://ecsource.jove.com/files/ftp_upload/54991/54991fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54991/54991fig4.jpg" /> <str<html>翁&gt;图4:PMEL17 CDR环聚焦肽残留PRO4，Val7和Thr8。 （A）中的YLE-9V肽和PMEL17 CDR环残基之间的联系方式的示意图（彩色编码如在图2A）。在面板的底部的数字显示了TCR与肽之间的总接触。 （B）中的PMEL17 TCR和所述YLE-9V肽（绿色棒）表示范德之间联系范德华接触（黑色虚线）和氢键（红色虚线）由TCRCDR3α（蓝色）制成，CDR1β（黄色） ，CDR2β（AQUA），和CDR3β（橙色）循环。在下部面板是YLE PRO4，Val7和Thr8，之间的触点的紧密视图分别和TCR CDR环残基（枝颜色编码为在图1A）。截止3.4的氢键和4对于范德华联系。从31参考授权转载。rge.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/54991/54991fig5.jpg" /> 图5:HLA-A * 0201主办YLE，YLE-3A的构象比较，A2-YLE-5A肽。 （A）YLE（深绿色棒）和YLE-3A（橙色支）由HLA-A的叠加* 0201α1螺旋（灰色卡通）肽对齐。盒装残基表明Glu3的突变成丙氨酸。该插图显示Glu3Ala替代如何导致一个位置的邻居残留PRO4在A2-YLE-3A结构相比，A2-YLE结构转变（黑色箭头）。 （B）YLE（深绿色棒）和YLE-5A（粉红色支）由HLA-A * 0201α1螺旋（灰色卡通）的叠加肽对齐。盒装残基表明甘氨酸5的突变成丙氨酸。从参考许可转载<sup&gt; 31。 <a href="http://ecsource.jove.com/files/ftp_upload/54991/54991fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> TCR </td><td> CDR1α </td><td> CDR2α </td><td> CDR3α </td><td> CDR1β </td><td> CDR1β </td><td> CDR1β </td></tr><tr><td> PMEL17 </td><td> DSAIYN </td><td> IQSSQRE </td><td> CAVLSSGGSNYKLTFG </td><td> SGHTA </td><td> FQGTGA </td><td> CASSFIGGTDTQYFG </td></tr><tr><td> GP100 </td><td> TSINN </td><td> IRSNERE </td><td> CATDGDTPLVFG </td><td> LNHDA </td><td> SQIVND </td><td> CASSIGGPYEQYFG </td></tr><tr><td><str翁&gt; MPD </td><td> KALYS </td><td> LLKGGEQ </td><td> CGTETNTGNQFYFG </td><td> SGHDY </td><td> FNNNVP </td><td> CASSLGRYNEQFFG </td></tr><tr><td> 296 </td><td> DSASNY </td><td> IRSNVGE </td><td> CAASTSGGTSYGKLTFG </td><td> MNHEY </td><td> SMNVEV </td><td> CASSLGSSYEQYFG </td></tr></tbody></table> 表1:PMEL17 的TCR CDR3地区，GP100，MPD和296具体的gp100，电阻温度系数的对齐。从31参考授权转载。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 肽序列 </td><td> 肽 </td><td> PMEL17 TCR TRAV21 TRBV7-3亲和杀敌 </td><td> GP100 TCR TRAV17 TRBV19阿菲无穷大杀敌 </td></tr><tr><td> YLEPGPVTA </td><td> YLE </td><td> 7.6±2微米</td><td> 26.5±2.3μM </td></tr><tr><td> YLEPGPVT V </td><td> YLE-9V </td><td> 6.3±1.2μM </td><td> 21.9±2.4μM </td></tr><tr><td> 一个 LEPGPVTA </td><td> YLE-1A </td><td> 15.9±4.1μM </td><td> 60.6±5.4μM </td></tr><tr><td> YL 一个 PGPVTA </td><td> YLE-3A </td><td>无约束力</td><td>无约束力</td></tr><tr><td> YLE 一个 GPVTA </td><td> YLE-4A </td><td> 19.7±1.3μM </td><td> 144.1±7.8μM </td></tr><tr><td> YLEP 一个 PVTA </td><td> YLE-5A </td><td> &gt; 1毫米</td><td> &gt; 1mM的</td></tr><tr><td> YLEPG 一个 VTA </td><td> YLE-6A </td><td> 11.4±2.7μM </td><td> 954.9±97.8μM </td></tR&gt; <tr><td> YLEPGP 一个 TA </td><td> YLE-7A </td><td> 31.1±4μM </td><td> 102.0±9.2μM </td></tr><tr><td> YLEPGPV A A </td><td> YLE-8A </td><td> 38.1±7.4μM </td><td> 121.0±7.5μM </td></tr></tbody></table> 表2:PMEL17 TCR的亲和力分析（K D）和GP100 TCR到的gp100 280-288肽变体。从31参考授权转载。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 参数 </td><td> PMEL17 TCR-A2-YLE-9V </td><td> A2-YLE </td><td> A2-YLE-3A </td><td> A2-YLE-5A </td></tr><tr><td> PDB代码&lt;/ STRONG&gt; </td><td> 5EU6 </td><td> 5EU3 </td><td> 5EU4 </td><td> 5EU5 </td></tr><tr><td> 数据统计 </td><td></td><td></td><td></td><td></td></tr><tr><td>空间群</td><td> P1 </td><td> P1 21 1 </td><td> P1 </td><td> P1 21 1 </td></tr><tr><td>晶胞参数（） </td><td> A = 45.52，B = 54.41，C = 112.12，A = 85.0°，B = 81.6°，G = 72.6° </td><td> A = 52.81，B = 80.37，C = 56.06，B = 112.8° </td><td> A = 56.08，B = 57.63，C = 79.93，A = 90.0°，B = 89.8°，G = 63.8° </td><td> A = 56.33，B = 79.64，C = 57.74，B = 116.2° </td></tr><tr><td>辐射源</td><td> DIAMOND I03 </td><td> DIAMOND I03 </td><td> DIAMOND I02 </td><td> DIAMOND I02 </td></tr><tr><td>波长（埃） </td><td> 0.9763 </td><td> 0.9999 </td><td> 0.9763 </td><td> 0.9763 </td></tr><tr><td> Measu红色的分辨率范围（A） </td><td> 51.87 - 2.02 </td><td> 45.25 - 1.97 </td><td> 43.39 - 2.12 </td><td> 43.42 - 1.54 </td></tr><tr><td>外分辨率壳牌（） </td><td> 2.07 - 2.02 </td><td> 2.02 - 1.97 </td><td> 2.18 - 2.12 </td><td> 1.58 - 154 </td></tr><tr><td>观察思考</td><td> 128191（8,955） </td><td> 99442（7056） </td><td> 99386（7,463） </td><td> 244577（17745） </td></tr><tr><td>独特思考</td><td> 64983（4785） </td><td> 30103（2249） </td><td> 49667（3,636） </td><td> 67308（4,962） </td></tr><tr><td>完备性（％） </td><td> 97.7（96.7） </td><td> 98.5（99.3） </td><td> 97.4（96.7） </td><td> 99.6（99.9） </td></tr><tr><td>多重</td><td> 2.0（1.9） </td><td> 3.3（3.1） </td><td> 2.0（2.1） </td><td> 3.6（3.6） </td></tr><tr><td> I /西格玛（I） </td><td> 5.5（1.9） </td><td> 7.2（1.9） </td><td> 6.7（2。3） </td><td> 13（2.3） </td></tr><tr><td> RPIM（％） </td><td> 5.7（39.8） </td><td> 8.8（44.7） </td><td> 8.7（41.6） </td><td> 4.5（35.4） </td></tr><tr><td> - [R 合并 （％） </td><td> 7.8（39.6） </td><td> 9.8（50.2） </td><td> 8.7（41.6） </td><td> 5.0（53.2） </td></tr><tr><td> 细化统计 </td><td></td><td></td><td></td><td></td></tr><tr><td>决议（A） </td><td> 2.02 </td><td> 1.97 </td><td> 2.12 </td><td> 1.54 </td></tr><tr><td>不使用反射</td><td> 61688 </td><td> 28557 </td><td> 47153 </td><td> 63875 </td></tr><tr><td>在Rfree集无反射</td><td> 3294 </td><td> 1526 </td><td> 2514 </td><td> 3406 </td></tr><tr><td> - [R cryst（不切断）（％） </td><td> 18.1 </td><td> 19.7 </td><td> 17.2 </td><td> 17.0 </td></tr><tr><td> r 无 </td><td> 22.2 </td><td> 25.5 </td><td> 21.1 </td><td> 20.1 </td></tr><tr><td> 根均理想几何偏差平方 </td><td></td><td></td><td></td><td></td></tr><tr><td>键长（埃） </td><td> 0.018（0.019）* </td><td> 0.019（0.019）* </td><td> 0.021（0.019）* </td><td> 0.018（0.019）* </td></tr><tr><td>键角（°） </td><td> 1.964（1.939）* </td><td> 1.961（1.926）* </td><td> 2.067（1.927）* </td><td> 1.914（1.936）* </td></tr><tr><td>整体坐标误差（） </td><td> 0.122 </td><td> 0.153 </td><td> 0.147 </td><td> .055 </td></tr><tr><td>拉马钱德兰统计</td><td></td><td></td><td></td><td></td></tr><tr><td>最惠国待遇</td><td> 791（96％） </td><td> 371（98％） </td><td> 749（99％） </td><td> 384（98％） </td></tr><tr><td>允许的</td><td> 32（4％） </td><td> 6（2％） </td><td> 10（1％） </td><td> 5（1％） </td></tr><tr><td>离群值</td><td> 2（0％） </td><td> 3（1％） </td><td> 1（0％） </td><td> 2（0％） </td></tr></tbody></table> 表3: 数据缩减和细化统计（分子置换）。从31参考授权转载。括号中的数值是分辨率最高的外壳。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> HLA /肽残基 </td><td> TCR残留 </td><td> 第范德华（≤4Å） </td><td> 第氢键（≤3.4Å） </td></tr><tr><td> Gly62 </td><td> αGly98 </td><td> 3 </td><td></td></tr><tr><td></td><td> αSer99 </td><td> 1 </td><td></td></tr><tr><td> Arg65 </td><td> αSer99 </td><td> 2 </td><tD&gt; </td></tr><tr><td> Arg65Ø </td><td> αAsn100Nδ2 </td><td> 2 </td><td> 1 </td></tr><tr><td> Arg65 NH1 </td><td> βAsp58Oδ2 </td><td></td><td> 1 </td></tr><tr><td></td><td> βSer59 </td><td> 8 </td><td></td></tr><tr><td> Lys66 </td><td> αGly98 </td><td> 1 </td><td></td></tr><tr><td></td><td> αSer99 </td><td> 4 </td><td></td></tr><tr><td></td><td> αAsn100 </td><td> 4 </td><td></td></tr><tr><td> Ala69 </td><td> αAsn100 </td><td> 2 </td><td></td></tr><tr><td></td><td> βAla56 </td><td> 2 </td><td></td></tr><tr><td> Gln72Nε2 </td><td> βGln51Ø </td><td> 3 </td><td> 1 </td></tr><tr><td></td><td> βGly54 </td><td> 7 </td><td></td></tr><tr><td></td><td> βAla55 </td><td> 1 </td><tD&gt; </td></tr><tr><td> Thr73 </td><td> βGln51 </td><td> 1 </td><td></td></tr><tr><td> Val76 </td><td> βGln51 </td><td> 3 </td><td></td></tr><tr><td></td><td> βGly52 </td><td> 2 </td><td></td></tr><tr><td> Lys146 </td><td> βPhe97 </td><td> 3 </td><td></td></tr><tr><td></td><td> βIle98 </td><td> 3 </td><td></td></tr><tr><td> Ala150 </td><td> βIle98 </td><td> 1 </td><td></td></tr><tr><td></td><td> βAsp102 </td><td> 3 </td><td></td></tr><tr><td> Val152 </td><td> βIle98 </td><td> 1 </td><td></td></tr><tr><td> Glu154 </td><td> αTyr32 </td><td> 1 </td><td></td></tr><tr><td> Gln155ñ </td><td> αTyr32OH </td><td> 4 </td><td> 1 </td></tr><tr><td> Gln155Oε1 </td><td> βThr101ñ </td><td> 10 </td><td> 1 </td></tr><tR&gt; <td> Tyr1 OH </td><td> αGly97Ø </td><td> 1 </td><td> 1 </td></tr><tr><td></td><td> αGly98 </td><td> 1 </td><td></td></tr><tr><td></td><td> αSer96 </td><td> 1 </td><td></td></tr><tr><td> Glu3 </td><td> αTyr101 </td><td> 1 </td><td></td></tr><tr><td> PRO4 </td><td> αSer96 </td><td> 1 </td><td></td></tr><tr><td></td><td> αSer99 </td><td> 1 </td><td></td></tr><tr><td></td><td> αAsn100 </td><td> 4 </td><td></td></tr><tr><td> ØPRO4 </td><td> αTyr101ñ </td><td> 14 </td><td> 1 </td></tr><tr><td> Gly5 </td><td> αTyr101 </td><td> 3 </td><td></td></tr><tr><td></td><td> βGly100 </td><td> 2 </td><td></td></tr><tr><td> Val7 </td><td> βIle98 </td><td> 7 </td><td></td></tr><tr><td></td><td> βGly99 </td><td> 2</td><td></td></tr><tr><td></td><td> βGly100 </td><td> 2 </td><td></td></tr><tr><td> Thr8 </td><td> βThr31 </td><td>五</td><td></td></tr><tr><td></td><td> βGln51 </td><td> 1 </td><td></td></tr><tr><td></td><td> βPhe97 </td><td> 1 </td><td></td></tr><tr><td> Thr8ñ </td><td> βIle98Ø </td><td> 6 </td><td> 1 </td></tr></tbody></table> 表4:PMEL17 TCR-A2-YLE-9V联系表。从31参考授权转载。

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Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

利用X射线晶体学，生物物理学，和功能分析，以确定管理机制T细胞癌抗原的受体识别

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

using-x-ray-crystallography-biophysics-functional-assays-to-determine

Research

JoVE Journal

Immunology and Infection

11.3K 次观看.  Cardiff University. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments. 

视频: Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

竞争的原点实验研究肠嗜T细胞迁移

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

鞘糖脂抗原的质谱分析

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

紧凑型超支化聚乙二醇接枝的膜抗原受保护的功能性红血细胞

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

该<em&gt;离体</em&gt;文化与模式识别小鼠肠道组织体受体刺激

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.